人白介素-2基因打靶载体的构建、转染及鉴定

Construction,transfection and identification of human IL-2 gene targeting vector

为研究人白介素-2基因在奶山羊胎儿成纤维细胞中的表达,构建人白介素-2基因的山羊β-酪蛋白位点打靶载体。以质粒pBC1为模板扩增山羊β-酪蛋白上游2.5 kb和下游3.5 kb的序列作为5′和3′同源臂,将克隆得到的人白介素-2基因和neo基因插入到2个同源臂之间,获得山羊β-酪蛋白基因打靶载体pBNI53。利用脂质体将重组质粒转染奶山羊胎儿成纤维细胞进行G418筛选,并采用PCR和实时定量PCR方法对人白介素-2基因在稳定株中的表达进行验证。检测结果表明,本试验构建的打靶载体pBNI53成功转染奶山羊胎儿成纤维细胞,将人白介素-2基因成功整合至奶山羊胎儿成纤维细胞基因组中,为通过体细胞核移植法制备人白介素-2基因乳腺生物反应器的研究奠定了基础。

Targeting vector for the human IL-2 gene knocking-in the goat p-casein locus was constructed to study the expression of the human IL-2 gene in fibroblasts of the fetal dairy goat. Goat p-casein gene targeting vector pBNI53 was obtain by cloned goat β-casein upstream 2.5 kb and downstream 3.5 kb sequence as the 5＇ and 3＇ homologous arm using pBC1 as a template,with inserted human IL-2 gene and neo gene. The plasmid was transient transfected into goat fetal fibroblasts by Iiposome, and then the transfected cells were sieved by G418, then in the stable lines the gene expression was detected by PCR and RT-PCR. After the detection by PCR and RT-PCR, the results suggested the target vector was constructed successfully,and the human IL-2 gene successfully integrated into the dairy goat fibroblasts, which lay a good foundation for the researching of producing human interleukin-2 gene in mammary gland bioreactor by somatic cell nuclear transfer.